The present invention in the field of biotechnology relates to a nucleic acid molecule. In particular, the present invention relates to a universal nucleic acid drug vector enhancing mRNA stability and translatability.
The nucleic acid drug applies DNA and RNA as a vaccine or a therapeutic drug for prevention and treatment of diseases in clinical applications. For many years, RNA has been considered to be unstable and susceptible to degradation. Thus most research in nucleic acid drugs, in particular nucleic acid vaccines, is based on DNA vaccines. A DNA vaccine is a plasmid DNA containing a foreign antigen gene sequence. It is delivered into the host body and enters the nucleus through the cellular and nuclear membranes. In the nucleus, the delivered foreign antigen gene DNA is transcribed into mRNA, which is then transported to the cytoplasm and translated into protein by ribosomes in the cytoplasm. The expressed protein can be taken up and processed by antigen presenting cells (APCs) such as dendritic cells (DCs) into multiple epitopes, which are bound with major histocompatibility complex (MHC) in animals or human leukocyte antigen (HLA) in human and presented to T cells, further eliciting immune responses such as generating cytotoxic T lymphocytes (CTLs) and antibodies, and achieving the purpose of prevention and treatment of diseases such as cancer and viral diseases. Also the transcribed mRNA can be used as therapeutic drugs.
Conventional DNA vaccine vectors include pcDNA3.1 and pVAX1. Among them, pcDNA3.1 is banned by US Food and Drug Administration (FDA) from human clinical use because pcDNA3.1 contains an ampicillin resistance gene. Since a DNA vaccine does not easily pass through the cellular and nuclear membranes, only a few DNA molecules can enter the nucleus, making it difficult to stimulate the body and further elicit a strong immune response. Therefore, no DNA vaccine has yet been approved for human clinical use. Currently, the employed electroporation method greatly improves the transfection efficiency and the immune effect of DNA vaccines, but there are still concerns regarding whether the plasmid DNA can be integrated into the host cell's genome.
In recent years, improvements in plasmid vectors have increased the stability of the in vitro transcribed mRNA, turning our attention to mRNA drugs, especially to mRNA vaccines. pGEM4Z/GFP/A64 and pGEM4Z/OVA/A64 are constructed based on pGEM4Z/A64 vector and made as templates for producing the in vitro transcribed mRNAs, which are inoculated via the intranasal route to induce anti-tumor immunity [Phua K K, et al. Sci Rep. 2014; 4:5128]. Using pcDNA3.1-64A and pSP73-Sph/A64, several vectors containing tumor-associated antigens (TAAs), glucocorticord-induced TNFR-related protein monoclonal antibody (GITR mAb) and cytotoxic T-lymphocyte-associated protein-4 mAb (CTLA-4 mAb) are respectively constructed and used for producing the corresponding in vitro transcribed mRNAs, which are electroporated into dendritic cells (DCs). Subsequently the obtained DC-mRNA vaccines are used for enhancing anti-tumor immunity [Pruitt S K, et al. Eur J Immunol. 2011; 41(12): 3553-63]. pSpjC-βglacZβgan and pT7TSiβggfpβgan are respectively constructed, resulting in LacZ and green fluorescent protein (GFP) genes flanked by 5′-untranslated region (UTR) and 3′UTR from Xenopus laevis β-globin respectively [Hoerr I, et al. Eur J Immunol. 2000; 30 (1): 1-7]. The plasmid vectors containing TAAs such as mucin1 (MUC1), carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (Her-2/neu), telomerase, survivin and melanoma-associated antigen 1 (MAGE-1) are respectively constructed utilizing pSP64-Poly (A)-EGFP-2 provided by V. F. I. Van Tendeloo and taken as templates for producing the in vitro transcribed mRNAs, which are used for anti-tumor immunity [Rittig S M, et al. Mol Ther. 2011; 19 (5): 990-9]. Also 5′top UTR is artificially synthesized and applied for increasing mRNA stability [Andreas Thess. US 20150050302 A1. Artificial nucleic acid molecules comprising a 5′top utr]. Several plasmids containing multiple mutant major histocompatibility complex (MHC) class II epitope sequences are respectively constructed using pST1-Sp-MITD-2hBgUTR-A120 and used for producing the in vitro transcribed mRNAs, which are inoculated into the body for generating personalized anti-cancer immunity [Kreiter S, et al. Nature 2015; 520 (7549): 692-6].
Among the above mentioned vectors, pGEM4Z/A64, pcDNA3.1-64A, pSP73-Sph/A64 and pSP64-Poly (A)-EGFP-2 do not have 5′UTR and 3′UTR, and contain only a short polyadenylation (poly A) tail (64A) so that the mRNA in vitro transcribed utilizing the above vectors is susceptible to degradation. Although containing 5′UTR and 3′UTR, pSpjC-βglacZβgan and pT7TSβggfβgan contain 3′UTR with only a Xenopus laevis β-globin so that their effect of stabilizing the in vitro transcribed mRNA is not ideal. pST1-Sp-MITD-2hBgUTR-A120 contains 3′UTR (with two β-globin) and poly A (120A), but it does not contain TTATT sequence as a terminator after poly A (120A) and its 5′UTR is not ideal. Therefore, there is still room for improvement. Other reported mRNA vaccine vectors that are not mentioned here are mostly made with minor improvements on the above plasmids.
Currently almost all the bacterial antibiotic resistance genes of plasmid vectors for generating the in vitro transcribed mRNA vaccines are ampicillin resistance genes. Before the in vitro transcribed mRNA can be deemed effective for human clinical use, it is necessary to check whether the ampicillin resistance gene remains in the final product. In addition, according to the provisions of the FDA, the plasmid vectors containing ampicillin resistance gene cannot be used as DNA vaccines for human clinical use.